Targeting TMEM205 mediated drug resistance in ovarian clear cell carcinoma using oncolytic virus

Background Ovarian clear cell carcinoma (OCCC) accounts for approximately 8–10% of epithelial ovarian cancers in the United States. Although it is rare, OCCC usually presents with treatment challenges and the overall prognosis is far worse than high grade serous ovarian cancer HGSOC. The objective of this study was to examine the therapeutic relevance of combining oncolytic virus with cisplatin for ovarian cancer clear cell carcinoma (OCCC). Results We identified that TMEM205, a recently discovered transmembrane protein, contributes to chemoresistance in OCCC cells via the exosomal pathway. Mechanistically, TMEM205 undergoes ligand-independent constitutive endocytosis and co-localizes with Rab11 to contribute to the late recycling endosomes in a clathrin-independent manner. Further, we observed that oncolytic virus (oHSV) pretreatment followed by treatment with cisplatin decreases TMEM205 expression and sensitizes cells to cisplatin in a synergistic manner in OCCC cells. TMEM205 interacts with glycoprotein-C of oHSV post-infection; both of these proteins undergo ubiquitination and ultimately get shuttled outside the cell via exosomes. Thus, we demonstrate the mechanotransduction pathway of TMEM205-mediated chemoresistance along with targeting this pathway using oHSV and cisplatin as a powerful therapeutic strategy for OCCC. oHSV combination with cisplatin inhibits OCCC tumor growth in vivo in immunodeficient and immunocompetent mice models. Conclusion Our results suggest that the combination of oHSV and cisplatin in immunocompetent as well as immune deficient OCCC tumor bearing mice reduces overall tumor burden as well as metastatic disease thereby providing survival benefit. Additionally, the detection of TMEM205 in exosomal cargo early in OCCC development has potential to be exploited as a biomarker. Supplementary Information The online version contains supplementary material available at 10.1186/s13048-022-01054-5.

Mouse ovarian cancer ascites derived from the ascites fluid collected from immunocompetent mice injected with ID8 cells. The MOCCs were cultured in medium and mixed with ID8 cells in a 1:1 ratio for injecting back into more immunocompetent mice. The resultant mice develop tumors within 4 weeks of injection in contrary to the 8-10 weeks time taken when injected with ID8 cells only OC tumor growth and metastasis in orthotopic mice, mimicking the clinical disease observed in patients with ovarian cancer.
Polyvinylidene fluoride (PVDF) membrane and molecular-weight markers were obtained from Bio-Rad (Hercules, CA). Antibodies, along with the seller information and dilutions, used in the current study have been listed in Sup. Division of Gynecologic oncology, Kyoto, Japan. These cells are very well characterized and published in ovarian clear cell carcinoma research 1-3 . These are even available for purchase with companies like ExPasy. Cells once thawed were used for only 3 months and we confirmed them for mycoplasma activity using ATCC ® Universal Mycoplasma Detection Kit, every 2 months.
Once the frozen cells were thawed, they were passaged for 5 times only and discarded thereafter and a fresh vial was thawed. Immunocytochemistry (ICC): Cells in RPMI medium were seeded onto sterile glass coverslips in 6-well plates with an average population of 50,000 cells/well. After 24 hours of culture the cells were washed, fixed, and incubated with primary antibody according to a previously described ICC protocol 4, 5 . For studies pertaining to Fig. 1 in "Expression of TMEM205 and its involvement in chemoresistance" section, the cells were not permeabilised using Tween-20 and PBS was used in all the steps, to maintain the membrane integrity.
For treatment with GFP labeled cisplatin (Michigan Diagnostics), OVTOKO or OV TM Si cells were treated with 10 µM of GFP labeled cisplatin for 6 hours followed by fixing and ICC.
For ICC of internalized TMEM205, OVTOKO cells were plated in 6 well plates with cover slips cells were labeled with anti-TMEM205 Ab for 20 min at 4°C and then incubated at 37°C for 15 min to allow for endocytosis To visualize only the internalized TMEM205, the epitope of the TMEM205 Ab on the surface was blocked by incubating the cells with unconjugated goat antirabbit IgG at 4°C. After fixation and permeabilization as described, internalized TMEM205 was labeled with Brilliant Violet conjugated Donkey anti-Rabbit IgG (H+L). Images were collected with FV3000 confocal laser scanning microscope using LSM Image Browser (Zeiss) software.
Antibodies used are listed in Table1.
Immunoblot analysis: Cell lysates were prepared in non-denaturing lysis buffer as previously described and subjected to immunoblot analyses 6, 7 . For the initial detection experiments, the SDS PAGE gel was stained with coomassie blue. The band appearing around 25kDa was excised and sent for protein identification to Prot Tech (Phoenixville, PA). Out of 23 proteins identified, the most abundant non contaminant protein was found to be TMEM205 and selected for this study.

Isolation of Membrane Fraction (MF)
We followed the Abcam protocol for subcellular fractionation of membrane proteins. Briefly, OVTOKO cells were lysed in subcellular fractionation buffer (250mM sucrose, 20mM HEPES, 10mM KCl, 1.5 mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT, PI cocktail). , homogenized through a 25G needle for 10 times and incubated on ise for 10 minutes. The pellet was centrifuged at 720G for 5 minutes and centrifuged again in fractionation buffer at 720G for 10 minutes. The pellet is the nuclear fraction (resuspended in standard lysis buffer) while supernatant was centrifuged at 10000G . the supernatant wa centrifuged in an ultracentrifuge at 100,000G for 1hour.
The resultant pellet was the membrane fraction which was re-suspended in the standard lysis buffer for the SDS PAGE gel and WB. The WB was probed with anti-TMEM205, EpCAM for confirming MF and with anti β-actin to confirm nuclear fraction. Antibody, clone ICR4 (EMD Millipore) in TBST for 18 hours, washed and probed with a rat secondary HRP antibody for 1 hour, followed by chemiluminescence based detection as described for the WB.
The pixel density of the resulting dots on the membrane was measured using Li-Cor software.
Each experiment was repeated 3 times in different sets. The g DNA was also run on a 0.75% agarose gel in 1X TAE and stained with ethidium bromide and pictured using a gel documentation system. OVTOKO control DNA digested with DNAse (1U) was loaded as positive control.

oHSV immunoprecipitation
For oHSV immunoprecipitation experiments, oHSV protein lysates were prepared in PBS without any detergent as previously described 12 . Cells were lysed in PBS containing protease inhibitors using acid washed glass beads (425-600µm, Sigma-Aldrich). oHSV particles @ MOI=1 were incubated with 100 µg of protein lysate for 12 h at 4˚C with constant gentle agitation. oHSV envelope glycoprotein antibodies (gC or gD) were added to the mixture and incubated for another 2 h at 4˚C. Pierce protein A/G magnetic beads (Thermo Fisher Scientific, Cat. No. 88802) were added to the mixture and incubated for another 4 h at 4˚C. Beads were washed 6 times with a wash buffer (20 mM HEPES, 200 mM NaCl, 1 mM EDTA containing 10 mg/ml BSA). One gel was stained with coomassie blue to extract band of interest and another one was blotted on a membrane and probed with anti TMEM205 or gC antibodies.
oHSV binding assay For the study of oHSV binding, OVTOKO or OV TM Si cells seeded in 6 well plates and incubated with oHSV in duplicates for 30 min on ice. One set was washed with PBS (3X5min) to remove unbound oHSV particles. The other set of cells were first trypsinized followed by washing with PBS, to remove the bound viral particles. Incubation of cells on ice allows viral binding but prevents viral entry. Total DNA was extracted as explained above and subsequently processed for quantification of viral DNA amount by quantitative PCR (qPCR). oHSV genome equivalents per cells was calculated by determining host cell number by amplification of the PI15 gene as described before 12 .
oHSV viral entry assay For viral entry assay, OVTOKO or OV TM Si cells were incubated with oHSV as mentioned above. The trypsinised as well as non trypsinised cells were subsequently transferred to a 37˚C incubator with fresh media. Both the cell types were grown for another 4 h for viral entry. Total DNA was extracted and used for quantification of viral DNA amount by qPCR 12 . Mice Core Lab as well as the immunocompetent mice.
The cell lines used are explained in Table 3.
Additionally, we used 2 kinds of female mice • Nude mice obtained from the OSU transgenic Facility • Immunocompetent mice C57BL/6J (Charles River Labs) A mixture of ID8 cells and mouse ascites derived cells (MOCCs) were mixed in a ratio of 1:1. The culture and growth of MADCs is explained in Table 3.
Tumor growth was monitored Statistical Analysis: Results were expressed as mean ± S.E. Comparisons between groups were made by the Student t-test for all the graphs. The significance level was set at p ≤ 0.05.